An anti-HER2 biparatopic antibody that induces unique HER2 clustering and complement-dependent cytotoxicity

Human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase that plays an oncogenic role in breast, gastric and other solid tumors. However, anti-HER2 therapies are only currently approved for the treatment of breast and gastric/gastric esophageal junction cancers and treatment resistance remains a problem. Here, we engineer an anti-HER2 IgG1 bispecific, biparatopic antibody (Ab), zanidatamab, with unique and enhanced functionalities compared to both trastuzumab and the combination of trastuzumab plus pertuzumab (tras + pert). Zanidatamab binds adjacent HER2 molecules in trans and initiates distinct HER2 reorganization, as shown by polarized cell surface HER2 caps and large HER2 clusters, not observed with trastuzumab or tras + pert. Moreover, zanidatamab, but not trastuzumab nor tras + pert, elicit potent complement-dependent cytotoxicity (CDC) against high HER2-expressing tumor cells in vitro. Zanidatamab also mediates HER2 internalization and downregulation, inhibition of both cell signaling and tumor growth, antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP), and also shows superior in vivo antitumor activity compared to tras + pert in a HER2-expressing xenograft model. Collectively, we show that zanidatamab has multiple and distinct mechanisms of action derived from the structural effects of biparatopic HER2 engagement.


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Life sciences study design
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Sample size
Data exclusions Replication Randomization

Blinding
The atomic models generated in this study have been deposited into the Protein Data Bank with accession number PDB 8FFJ. The corresponding cryo-EM density maps generated in this study have been deposited into the Electron Microscopy Data Bank with accession numbers EMD-29044. Other atomic models used in this study are available with accession numbers 1N8Z, 1S78, 6OGE. The remaining data are available within the Article, Supplementary Information, Source Data file or available from the corresponding author upon reasonable request. Source data are provided with this paper.
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A calculation of sample size by power analysis was used to determine animal numbers for in vivo studies that would allow for robust statistical analysis. Here, sample size estimation was performed using a pilot dataset from which variance parameters and effect size are approximated by fitting a mixed effects model. Using this model, data was simulated by sampling the fitted distributions multiple times across a range of different sample sizes and effect sizes. For each simulated dataset, the hypothesis test procedure was performed to produce a p-value, and the power is calculated as the proportion of simulations for which the p-value is below 0.05. A sample size was chosen that satisfied a desired effect size for a given study at a power of 90%. For all other studies, no sample-size calculations were performed. Sample size was determined to be adequate based on the magnitude and consistency of measurable differences between groups.
Data exclusions for the SPR data (Fig 2a and Fig S3b) are described in the methods and follow standard procedures for acceptance of model fitting for kinetic values. Kinetic values that had chi-square values > 10% of the maximum binding capacity (Rmax), U-values > 25 or tc values (i.e. the flow rate-independent component values, a modification of the mass transfer constant) in the range of 10E7 -10E9 with low SE (indicating mass transport) were excluded from the linear regression analysis. The experiment in Fig 2a is focused on measuring discrete changes in kd (off-rate) over low to high captured antibody surface densities on the chip, thus stringent acceptance criteria were applied to fitted kd (off-rate), ka (on-rate) values and subsequent Kd values. One NCI-N87 CDC experiment was not included in the n=6 data plot in figure 4a because this experiment tested a different concentration range compared to the other n=6 experiments; however, the fitted max effect data are included in the n=7 meta analysis in Table 1.
For the majority of experiments a minimum of 3 experimental replicates were performed. For supplementary experiments, a minimum of 2 and typically 3 experiments were performed. All attempts are replication were successful, with one exception. Two of three PBMC donor showed ADCC activity in JIMT-1 cells, one donor was inactive with all anti-HER2 Abs tested in JIMT-1 cells.
Animals in the in vivo study were randomly assigned to the blinded treatment groups. For all other studies, samples were allocated to experimental group without any previous selection.
For in vivo studies, samples were blinded to the experimentalists performing the work, measurements and initial data analysis.

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We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. A list with all antibodies used and antibody source and dilution is provided in Supplementary Table 11. Validation to human protein target was performed following variable strategies: cellular localization, molecular weight of bands in western blotting experiments; binding to recombinant target protein by ELISA, and control tissues, either by vendor and/or herein. Cell lines were authenticated by supplier and cell lines were used from source and expanded for a maximum of 20 passages.
Cell lines were routinely spot check tested for mycoplamsa, all tests were negative.
MCF7 and ZR-75-1 were used as representative HER2-low cell lines. MCF7 was used for on cell binding and complement dependent cytotoxicity (CDC) assays and ZR-75-1 cell line was used in the CDC assay. Data and conclusions from the MCF7 and ZR-75-1 cell lines are supported with data from other HER2-low cell lines including JIMT-1 and MDA-MB-175-VII.
Five to seven week old female athymic nude mice (NMRI-Foxn1nu, Harlan Laboratories) were used in the GXA 3054 PDX in vivo study. Six to eight week female BALB/c nude mice were used in the NCI-N87 xenograft in vivo study. Mouse strain serum was collected from six to eight week old male and female Balb/c, and male and female CB17 SCID.
The study did not involve wild animals.
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The study did not involve samples collected from the field.
All animal studies were carried out in accordance with all applicable international, national, and local laws and guidelines and protocols approved by the regional council Committee of Ethics of Animal Experts (Oncotest GmBh) and Institutional Animal Care and

March 2021
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Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Methodology
Sample preparation Instrument Software Cell population abundance Use Committee (CrownBio). Mouse strain serum (male and female Balb/c, male and female CB17 SCID) was collected under protocols approved by the Animal Care Committee at University of British Columbia.
On-cell binding assays: The target tumor cells at 40,000 cells/well were incubated in FACS buffer (PBS + 2% FBS) with primary antibodies for 1 h at 4°C. Cells were washed twice with FACS buffer and incubated with a secondary anti-human IgG antibody for 1 hr at 4°C. Following incubation, cells were washed with FACS buffer.
C1q and C3 fragment binding assays: C1q and C3 binding: The target tumor cells at 50,000/well were washed twice in serum free media and incubated in FACS buffer in the presence of pooled normal human serum and anti-HER2 antibodies for 15 min at 37°C. The target tumor cells were then incubated in FACS buffer (PBS + 2% FBS) with primary antibodies for 45 min at 4°C. Cells were then washed twice with FACS buffer.
CDC assays: The target tumor cells were incubated in FACS buffer in the presence of pooled normal human serum and anti-HER2 antibodies for 3 h at 37°C. Cells were stained with Propidium Iodide.
ADCC Assays: Human PBMCs were rested overnight in RPMI + 10% ultra low IgG FBS + 100 U/mL recombinant human IL-2 at 37°C. The following day, target tumor cells were stained with CMFDA and incubated with the rested PBMCs at an effector to target ratio of 5:1 and anti-HER2 antibodies for 4 h at 37°C. Cells were washed twice in FACS buffer and stained for viability with LIVE/DEAD Fixable Violet Dead Cell Stain.
ADCP Assays: Human monocytes were differentiated into macrophages by culturing human PBMCs in RPMI + 10% FBS + 10 ng/mL MCSF for 8 days. Macrophages were lifted and stained with Cytolight Rapid Red and co-cultured with tumor target cells stained with CellTracker Green CMFDA at an effector to target ratio of 2:1 for 1.5 h at 37°C. Cells were stained for viability using LIVE/DEAD Fixable Violet Dead Cell Stain.
Internalization Assays: Primary antibodies were coupled to anti-human IgG Fab fragment, covalently conjugated to AF488 fluorophore, at a 1:1 molar ratio for 24 h at 4°C. Target tumor cells were incubated in culture media with primary antibodies coupled to Fab-AF488 for 24 h at 37°C and 15 min at 4°C. Following incubation, cells were dissociated and washed with FACS buffer (PBS + 2% FBS). For a subset of samples, surface AF488 fluorescence was quenched with an anti-AF488 antibody for 45 min at 4°C.
HER2 Surface Downregulation Assays: Target tumor cells were seeded in 48-well plates at 50,000 cells/well. Next day, cells were treated with antibody for 24 hrs at 37°C. Following incubation, cells were harvested and stained with a non-competing, fluorescently labeled anti-HER2 antibody AF647 conjugate for 45 min at 4°C. Following incubation, cells were dissociated and washed with FACS buffer (PBS + 2% FBS).
Receptor Quantification Assays: Target tumor cells were seeded in 96-well plates at 50,000 cells/well. One drop of quantification bead sets 1-5 were added to separate eppendorf tubes. Target tumor cells were prepared with a violet viability stain and stained with or without a saturating level of fluorescently labeled anti-HER2 antibody AF647 conjugate and violet viability stain. Similarly, quantification beads sets 1-4 were incubated with anti-HER2 antibody AF647 conjugate and violet viability stain. Quantification bead set 5 (blank) was prepared in FACS buffer (PBS + 2% FBS) only. All samples were incubated for 30 min on ice and subsequently washed with FACS buffer.
BD LSRFortessa X-20, Sartorius Intellicyte iQue3 BD FACSDiva v9, Forecyt, FlowJo v10 All flow cytometry analysis was done on pure cultures of individual cell lines, not mixed cell populations. No statements are made about population abundance.
On cell Ab, C1q and C3 fragment binding: In all the experiments, a control sample lacking primary antibody was included and at least 2,000 events were acquired.